This relates to the fields of analytical chemistry and immunology and more specifically relates to an integrated assay device for determining the presence or amount of an analyte in a sample.
Assay technology involves methods or instrumentation for the detection or quantitation of one or more antigens or analytes in a sample. Immunoassays are based on the highly specific binding reaction between an antibody, or analyte receptor, and an antigen recognized by the antibody or antigen receptor. Antibodies are binding proteins produced by the immune system of vertebrates in response to substances identified by the immune system as foreign. Immunoassays are commonly used by the medical community to determine the presence, amount or identity of analyte in a biological sample for purposes such as diagnosis and for monitoring therapy. Immunoassays are also used for the detection of environmental contaminants. More recently, immunoassays have been used by non-technical persons in the home for private determinations of medical conditions such as pregnancy and ovulation.
Various approaches for performing homogeneous or heterogeneous immunoassays in both competitive and noncompetitive formats have been described in the literature. Homogeneous immunoassays are performed by combining labeled reagent with a sample and detecting labeled analyte without a separation step. Although homogeneous immunoassays are easy to perform, they are subject to matrix interference. In homogeneous assays it is difficult to ascertain the proportion of bound and free label in the reaction mixture, and thus sophisticated instrumentation is often required to detect and analyze the results. Heterogeneous immunoassay methods contain a separation step in which bound label is separated from free label.
The original enzyme linked immunosorbent assay (ELISA) methods were xe2x80x9ccompetitivexe2x80x9d heterogeneous assays in which an enzyme-labeled antigen or antibody competed with an antigen or antibody to be detected for a reaction site on a bead, pad or surface to which one member of an immunologically-coupling pair was attached. Subsequently, the xe2x80x9csandwichxe2x80x9d assay, a non-competitive assay, was developed. Non-competitive assays generally utilize antibodies in substantial excess over the concentration of analyte to be determined in the assay. In the sandwich assay, the antibody or antigen to be determined is xe2x80x9csandwichedxe2x80x9d by an immunochemical reaction between a solid surface treated with an immunological species reactive with the species to be determined and the same or a different reactive immunological species which had been coupled to a signal-generating label.
Competitive assays generally include a sample suspected of containing analyte, an analyte analog-assay conjugate, and the competition between these components for a limited number of binding sites on the antibody. Due to competition between unbound analyte and analyte analog-assay conjugate for analyte-receptor binding sites, as the analyte concentration increases, the amount of unbound analyte analog-enzyme conjugate increases, thereby decreasing the observed signal associated with the solid phase. The product of the enzyme reaction may then be measured using an instrument such as a spectrophotometer.
Exemplary analytes detected by immunoassays include haptens, hormones, peptides, proteins, deoxyribonucleic. acid (DNA), ribonucleic acids (RNA), metabolites of these materials and other substances of either natural or synthetic origin, which may be of diagnostic interest. Binding assays are generally useful for the in vitro determination of the presence and concentration of analyte in body fluids, food products, animal fluids, and environmental samples, such as the determination of specific hormones, peptides, proteins, therapeutic drugs, forensics, paternity and toxic drugs in human or animal blood or urine.
Numerous detection systems have been developed to detect or measure antibody-analyte complexes including enzyme-catalyzed chromogenic reactions, radionuclides, chemiluminescence, bioluminescence, fluorescence, fluorescence polarization and a variety of potentiometric and optical biosensor techniques.
One disadvantage to the presently available immunoassay methods is that multiple manipulations are often required by the individual performing the assay as reagents are added, mixed, incubated, separated and detected, thereby introducing the potential for error. Another disadvantage is that conventional immunoassays require a substantial amount of sample, which could be unavailable, difficult or painful to obtain. In addition, the accuracy of many immunoassays depends on precise sample and reagent measurements and the standardization of conditions such as incubation time and temperature.
Although some simplified immunoassays are available for use by non-technical personnel, these assays lack precision and provide the user with only a xe2x80x9cpositivexe2x80x9d or xe2x80x9cnegativexe2x80x9d response, no quantifiable results are produced. Furthermore, these presently available devices are composed entirely of membranes or absorbent fibrous materials that vary from lot to lot and contain imprecise volumes of sample and the reagents employed.
There is a continuing need for simple, rapid assays for the qualitative, semi-quantitative, and quantitative determination of analytes in a sample. In many situations, such assays need to be simple enough to be performed and interpreted by non-technical users outside of a laboratory.
Thus, simple assay methods are needed that will provide reliable, accurate and rapid results within and outside of conventional laboratory facilities in places such as hospitals, medical offices, homes, on the streets and in the field. There is also a great need for simple, inexpensive and easy-to-use assay devices, particularly immunoassay devices, that are easily manufactured and can be used by technical and non-technical personnel, such as emergency medical technicians, police, firefighters, corrections facilities, and military personnel.
Assay devices for the detection of analyte in a sample and methods for performing an assay using the devices are provided herein. Also provided are methods of manufacturing the assay devices. The devices can be used for nucleic acid-based assays, chemical assays, and immunoassays, including heterogeneous immunoassays for both competitive and sandwich immunoassay formats. Also provided are simple immunochromatographic strip detection membrane formats having increased precision and accuracy over existing formats.
The device includes self-contained, integrated components for conducting an assay for analyte with minimal manipulation by the individual performing the assay. Upon adding the sample to be analyzed and introducing a force to initiate the assay, no further interaction is required for assay completion. Therefore, by using the device, the assay can be performed by technical and non-technical personnel within and outside of a conventional laboratory environment. The components are miniaturized and compacted into a conveniently-sized, self-contained housing, thereby utilizing a minimal amount of space to facilitate transport and use.
The device is a continuous liquid flow channel having a proximal and a distal end, wherein the sample is introduced to the channel via a sample delivery means so that the sample travels toward the distal end of the channel to a detection membrane in fluid communication with the distal end of the channel. Continuous with the liquid flow channel, are a sample delivery means, one or more reservoirs containing one or more buffers and reagents necessary for conducting the assay, and, optionally, mixing or incubation reservoirs for combining the sample and reagents. The locations of the sample valve and reservoirs, and liquid volume capacities of the flow channel, sample delivery means and reservoirs can be modified and rearranged as needed to optimize the conditions for a wide variety of assay formats and analytes to be detected.
One or more liquid flow channels may be contained within a single housing for simultaneous or consecutive sample analysis.
The assay device optionally includes an initiating means, such as a pump, at the proximal end of the flow channel for initiating the flow of liquid through the liquid flow channel of the device. Reagents, including one or more labeled reagents, are contained within the liquid flow channel either in a liquid or lyophilized state.
The dimensions and geometric shape of the liquid flow channel, including the reagent reservoirs and mixing chambers, regulates the flow rate of the liquids through the channel, thereby controlling incubation, mixing or reaction time. The preferred internal, cross-sectional geometric shape of the liquid flow channel preferably contains at least one angle and is composed of one or more flat surfaces joined at one or more angles, such as a teardrop, pie-shape, triangle, trapezoid, square, rhombus, pentagon, hexagon, etc., rather than merely a continuous internal curved surface such as a circle or oval. Combinations of flat and curved surfaces are also contemplated in the assay device.
The device further includes sample delivery means for introducing a precise, predetermined volume of sample into the liquid flow channel. The sample delivery means optionally contains filtration means for separating interfering substances, such as the cellular components of the sample, before the sample is introduced into the flow of the liquid flow channel.
Buffer is useful for diluting out matrix effects, diluting samples to be within concentration range of detection means, and providing hydrostatic pressure to drive fluid flow. An assay buffer reservoir, located upstream from the location in the liquid flow channel where the sample is introduced by the sample delivery means, is useful for rinsing all of the sample out of the sample delivery means, thereby ensuring that the volume of sample introduced is precisely the predetermined volume.
As an additional option, the device further includes means for separating mobile reagents from immobilized reagents bound to a solid phase substance, such as a solid phase particle.
The detection membrane of the preferred device is a substrate upon which is immobilized means for detecting the labeled reagent that has reacted either directly or indirectly with analyte in the sample. Detection of label can be visual or with the aid of a detector known in the art for the detection of signal produced in an assay reaction, such as a spectrophotometer or reflectometer. The amount of label detected reflects the amount of analyte in the sample being analyzed. Additional reagents are optionally included to calibrate the device or monitor device performance or assay progress, including completion.
The assay method utilizing the device is useful for the detection of a wide variety of analytes including, but not limited to, environmental contaminant analytes, agricultural products, industrial chemicals, water treatment polymers, pharmaceutical drugs, drugs of abuse, and biological analytes, such as antigenic determinants of proteins, polysaccharides, glycoproteins, lipoproteins, nucleic acids and hormones, of organisms such as viruses, bacteria, fungi, parasites, plants and animals, including humans. The assay method is chemical in nature, and the preferred assay method is an immunoassay.
The device is manufactured by adding precise volumes of liquid reagents to predetermined regions of the liquid flow channel either prior to encapsulation of the channel or through apertures from the exterior of the housing to the channel that are subsequently sealed. The reagents may be lyophilized, or otherwise dried, to preserve activity or facilitate immobilization during transportation or storage.
It is therefore an object of the present invention to provide an assay device for immunoassays, nucleic acid-based assays and chemical assays that is self-contained and automatically executes all the steps of a multi-step assay through the use of precise control of the geometry of microfluidic channels and chambers etched or molded in the assay device housing material.
It is a further object of the present invention to provide an integrated means for delivering a precise, predetermined volume of sample to an assay device.
It is a further object of the present invention to provide an integrated means for preparing or processing a sample upon introduction of the sample to an assay device such as by filtration or extraction, thereby eliminating the need to process the sample.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample that can be used to detect a wide variety of analytes.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample that is simple, inexpensive, portable and user-friendly and can be utilized successfully by non-scientific personnel.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample that provides reliable, reproducible results on-site, in the home, or in the field.
It is a further object of the present invention to provide an assay device and method for the unattended detection of analyte after introduction of sample to the device.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample that requires no critical timing events or mixing of reagents by the individual performing the assay because all incubation times are determined by the dimensions of the device, such as the capillary channel geometry.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample without requiring precise sample or reagent measurements by the individual performing the assay.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample that provides results that are very accurate, very reproducible and highly controlled.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample that produces rapid results.
It is a further object of the present invention to provide a flexible assay device and method that can be adapted to various samples, assay formats and detection parameters.
It is a further object of the present invention to provide an assay device and method for the detection of analyte in a sample that produces results that can be analyzed by the naked eye in the absence of, or with minimal, instrumentation or training of the individual performing the assay.
It is a further object of the present invention to provide an assay device and method that is easily and inexpensively manufactured.
It is a further object of the present invention to provide assay and immunochromatographic strip detection membrane formats that provide for precise and accurate detection of analyte in a sample.